Elsevier

Experimental Cell Research

Volume 315, Issue 5, 10 March 2009, Pages 849-862
Experimental Cell Research

Research Article
The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation

https://doi.org/10.1016/j.yexcr.2008.12.023Get rights and content

Abstract

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

Introduction

Endogenous retroviruses are genetic remnants of ancestral retroviral infections of the germline with a characteristic ability to integrate their genomes into the host cell DNA as an essential part of their replication cycle [1], [2]. The human genome comprises up to 8% retroviral sequences [3], classified in 30–50 Human Endogenous Retrovirus (HERVs) families [4], most of which are defective, due to multiple mutations or deletions, and are therefore unable to retrotranspose [5]. HERVs are poorly expressed in normal somatic tissues, although they can act as inducible genetic elements and are transcriptionally up-regulated in response to both exogenous (e.g chemicals, UV radiations) [6], [7] and endogenous (e.g. cytokines, hormones) factors [6], [8], [9]. More recently, microarray analysis has shown that HERVs are constitutively and ubiquitously expressed and that their expression levels are developmentally regulated in both somatic and germline tissues [10], [11], [12]. In contrast with the traditional view that retroelements represent mere parasitic burdens of eukaryotic genomes, increasing data support the view that sub-populations of biologically active HERV families play roles both in physiological and pathological processes, by either providing functional RNA transcripts and/or proteins, or by generating mutations through retrotransposal integration events [for a review see 13].

Among other human endogenous retroviral families, the HERV-K family is the most recently amplified family integrated into the human genome only  <  5 million years ago and is thought to be preferentially associated with human diseases, being composed of biologically active retroelements that display polymorphic integration events within the human population [14], [15], [16], [17]. Consistent with these features, HERV-K is the only known retroviral family that has retained functional full-length open reading frames coding for the structural and enzymatic proteins gag, prt, pol and env [14], [18], [19]. More specifically, expression of the HERV-K family is linked to a variety of cancer types, including germ-cell [20], breast [21], ovarian [22] and haematological cancers [23], [24], and, more recently, melanoma, in which increased abundance of HERV-K-encoded proteins was detected both in primary skin tumours and in lymphatic metastases [19], [25]. HERV-K genes have indeed been found to be expressed in melanoma cell lines and primary melanoma cells. Furthermore, HERV-K-encoded proteins were revealed in touch preparations from patients with primary or metastatic melanoma as well as in melanoma biopsy-derived cell lines, but not in benign melanocytic lesions [19], [25]. Interestingly, HERV-K-derived virus-like particles are also typical of cell lines derived from teratocarcinoma [26], [27], breast cancer [28], melanoma [19], [25] and also of certain non-pathological embryonic tissues such as human placenta [29]. Together these lines of evidence converge to suggest an intriguing correlation between the development of human melanoma and HERV-K activity.

Here we show that human melanoma cell lines undergo a transition from an adherent to a non-adherent growth phenotype; this is accompanied by a variety of morphological and molecular alterations, typical of highly malignant cells. These changes, initially observed as a spontaneous occurrence in cell cultures, can also be induced experimentally by exposing melanoma cells to starvation conditions. These changes are related to massive production of HERV-K-related viral-like particles and are dependent on HERV-K expression, as revealed by the reversal of these features when HERV-K expression was deliberately down-regulated by RNA interference. As a whole, these data strongly point to activation of HERV-K expression as a key element – though not necessarily the only one – contributing to morphological and functional cell changes during melanoma progression.

Section snippets

Cells and cultures

M14 and A-375 human melanoma cells, Caco-2 and WiDr human colon adenocarcinoma cells, K-562 human chronic myelogenous leukaemia cells and Jurkat human acute T cell leukaemia cells were originally obtained from the American Type Culture Collection (ATCC, Rockville, MD). The human melanoma cell lines TVM-A12 and TVM-A197 were established in our laboratory as monolayers from two melanoma lesions of the same patient, obtained at the presentation and at the terminal stage of the disease,

Cell clones derived from a human melanoma cell line spontaneously develop altered growth and morphological features

Two cell clones were isolated by limiting dilution technique from the TVM-A12 human melanoma cell line (Fig. 1A) [30]. Cells of both clones grow as adherent monolayers (termed Clonead, Fig. 1B) and, as shown by scanning electron microscopy, display a characteristic polygonal shape and microvillous structures (Fig. 1E) that were more abundant compared to the parental cell line (Fig. 1D). In both cultures, we observed that, at passage 22 after cloning, some rounded villous cells passed

Discussion

The present results show that human melanoma cell lines undergo a transition from an adherent to a non-adherent growth phenotype, accompanied by morphological, immune and proliferative features typically associated with increased malignancy. We initially observed this transition as a spontaneously occurring event in subclones of the TVM-A12 melanoma cell line, derived from a primary melanoma lesion in our laboratory. The similarity between this phenotypic transition and the classical

Acknowledgments

The authors would like to thank Dr. Patrizia Lavia for her critical reading of the manuscript, Federica Andreola, Manuela Zonfrillo and Fabio Falcione for their precious technical assistance, Martino Tony Miele for his excellent secretarial assistance and Dr. Paolo Daniele Siviero for advice on Intellectual Property Right.

This work was supported by a grant from the Italian Ministry of Education, University and Research, Research Project of National Interest, to E. Garaci.

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